PROJECT SUMMARY The foundational mechanism of translation initiation was discovered in E. coli over 40 years ago. Despite the textbook perception that this mechanism is ubiquitous among all bacteria, E. coli is actually highly adapted to utilize Shine-Dalgarno sequences for translation initiation. Examination of genomic data across species reveals that fewer than half of all bacterial messages have Shine-Dalgarno sequences to position ribosomes at the initiation codon. Indeed, many pathogenic bacteria lack Shine-Dalgarno sites in most messages and the molecular mechanisms of start codon selection are largely unknown in these species. C. crescentus is currently the best model system to study non-Shine-Dalgarno translation initiation as it has a rapid doubling time, a broad array of genetic tools, and a low fraction of genes initiated at Shine-Dalgarno sites (23.5%), including 375 leaderless mRNAs containing a 5? AUG. By combining in vitro translation assays and in vivo ribosome profiling experiments we seek to define the important aspects of non-Shine-Dalgarno initiation in this organism. We believe that many of the characteristics of C. crescentus translation initiation will be shared with the translation initiation pathways of pathogenic organisms, potentially leading to the identification of new antibiotic targets.